The present invention relates generally to signaling molecules, specifically to signaling and mediator molecules in the hedgehog (Hh) cascade which are involved in cell proliferation and differentiation.
Development of multicellular organisms depends, at least in part, on mechanisms which specify, direct or maintain positional information to pattern cells, tissues, or organs. Various secreted signaling molecules, such as members of the transforming growth factor-beta (TGF-xcex2), Wnt, fibroblast growth factors and hedgehog families have been associated with patterning activity of different cells and structures in Drosophila as well as in vertebrates. Perrimon, Cell: 80: 517-520 (1995).
Segment polarity genes were first discovered in Drosophila, which when mutated caused a change in the pattern of structures of the body segments. These changes affected the pattern along the head to tail axis. Hedgehog (Hh) was first identified as a segment-polarity gene by a genetic screen in Drosophila melanogaster, Nusslein-Volhard et al., Roux. Arch. Dev. Biol. 193: 267-282 (1984), that plays a wide variety of developmental functions. Perrimon, supra. Although only one Drosophila Hh gene has been identified, three mammalian Hh homologues have been isolated: Sonic Hh (Shh), Desert Hh (Dhh) and Indian Hh (Ihh), Echelard et al., Cell 75: 1417-30 (1993); Riddle et al., Cell 75: 1401-16 (1993). Shh is expressed at high level in the notochord and floor plate of developing vertebrate embryos, and acts to establish cell fate in the developing limb, somites and neural tube. In vitro explant assays as well as ectopic expression of Shh in transgenic animals show that SHh plays a key role in neural tube patterning, Echelard et al. (1993), supra.; Ericson et al., Cell 81: 747-56 (1995); Marti, et al., Nature 375: 322-5 (1995); Roelink et al. (1995), supra; Hynes et al, Neuron 19: 15-26 (1997). Hh also plays a role in the development of limbs (Krauss et al., Cell 75: 1431-44 (1993); Laufer et al., Cell 79, 993-1003 (1994)), somites (Fan and Tessier-Lavigne, Cell 79, 1175-86 (1994); Johnson et al., Cell 79: 1165-73 (1994)), lungs (Bellusci et al., Develop. 124: 53-63 (1997) and skin (Oro et al., Science 276: 817-21 (1997). Likewise, Ihh and Dhh are involved in bone, gut and germinal cell development, Apelqvist et al., Curr. Biol. 7: 801-4 (1997); Bellusci et al., Dev. Suppl. 124: 53-63 (1997); Bitgood et al., Curr. Biol. 6: 298-304 (1996); Roberts et al., Development 121: 3163-74 (1995). Specifically, Ihh has been implicated in chondrocyte development [Vortkamp, A. et al., Science 273: 613-22 (1996)] while Dhh plays a key role in testis development. Bitgood et al., supra. With the exception of the gut, in which both Ihh and Shh are expressed, the expression patterns of the hedgehog family members do not overlap. Bitgood et al., supra.
At the cell surface, Hh function appears to be mediated by a multicomponent receptor complex involving patched (e.g., Ptch) and Smoothened (e.g., Smo), two multi-transmembrane proteins initially identified as segment polarity genes in Drosophila and later characterized in vertebrates. Nakano et al., Nature 341: 508-513 (1989); Goodrich et al., Genes Dev. 10: 301-312 (1996); Marigo et al., Develop. 122: 1225-1233 (1996); van den Heuvel, M. and Ingham, P. W., Nature 382: 547-551 (1996); Alcedo, J. et al., Cell 86: 221-232 (1996); Stone, D. M. et al., Nature 384: 129-34 (1996). Upon binding of Hh to Patched, the normal inhibitory effect of Patched on Smo is relieved, allowing Smo to transduce the Hh signal across the plasma membrane. It remains to be established if the Patched/Smo receptor complex mediates the action of all 3 mammalian hedgehogs or if specific components exist. Interestingly, a second murine Patched gene, Patched-2 was recently isolated [Motoyama, J. et al., Nature Genetics 18: 104-106 (1998)], but its function as a Hh receptor has not been established. In order to characterize Patched-2 and compare it to Patched with respect to the biological function of the various Hh, family members, Applicants have isolated the human Patched-2 gene. Biochemical analysis of Patched and Patched-2 show that both bind to all members of the Hh family with similar affinity and that both molecules can form a complex with Smo. However, the expression patterns of Patched-2 and Patched do not overlap. While Patched is expressed throughout the mouse embryo, Patched-2 is found mainly in spermatocytes which require Desert Hedgehog (Dhh) for proper development suggesting that Patched-2 mediates Dhh""s activity in the testis. Chromosomal localization of Patched-2 places it on chromosome 1p33-34, a region deleted in some germ cell tumors, raising the possibility that Patched-2 may be a tumor suppressor in Dhh target cells.
In one embodiment, the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity to (a) a DNA molecule encoding a patched-2 polypeptide comprising the sequence of amino acids 1 to 1203 of FIG. 1 (SEQ ID NO:2), or (b) the complement of the DNA molecule of (a); and encoding a polypeptide having patched-2 biological activity. The sequence identity preferably is  greater than 91%, more preferably about 92%, most preferably about 95%. In one aspect, the isolated nucleic acid has at least  greater than 91 %, preferably at least about 92%, and even more preferably at least about 95% sequence identity with a polypeptide having amino acid residues 1 to about 1203 of FIG. 1 (SEQ ID NO:2). In a further aspect, the isolated nucleic acid molecule comprises DNA encoding a human patched-2 polypeptide having amino acid residues 1 to about 1203 of FIG. 1. In yet another aspect, the invention provides for an isolated nucleic acid comprising DNA having at least a 95% sequence identity to (a) a DNA molecule encoding the same mature polypeptide encoded by the cDNA in ATCC Deposit No. 209778 (designation: pRK7.hptc2.Flag-1405), alternatively the coding sequence of clone pRK7.hptc2.Flag-1405, deposited under accession number ATCC 209778. In a still further aspect, the invention provides for a nucleic acid comprising human patched-2 encoding sequence of the cDNA in ATCC Deposit No. 209778 (designation: pRK7.hptc2.Flag-1405) or a sequence which hybridizes thereto under stringent conditions.
In another embodiment, the invention provides a vector comprising DNA encoding a human patched-2 polypeptide. A host cell comprising such a vector is also provided. By way of example, the host cells may be mammalian cells, (e.g. CHO cells), prokaryotic cells (e.g., E. coli) or yeast cells (e.g., Saccharomyces cerevisiae). A process for producing patched-2 polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of patched-2 and recovering the same from the cell culture.
In yet another embodiment, the invention provides an isolated patched-2 polypeptide. In particular, the invention provides isolated native sequence patched-2 polypeptide, which in one embodiment is a human patched-2 including an amino acid sequence comprising residues 1 to about 1203 of FIG. 1 (SEQ ID NO:2). Human patched-2 polypeptides with or without the initiating methionine are specifically included. Alternatively, the invention provides a human patched-2 polypeptide encoded by the nucleic acid deposited under accession number ATCC 209778.
In yet another embodiment, the invention provides chimeric molecules comprising a patched-2 polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a patched-2 polypeptide fused to an epitope tag sequence or a constant region of an immunoglobulin.
In yet another embodiment, the invention provides expressed sequence tag (EST) comprising the nucleotide sequences identified in FIG. 2A (905531) (SEQ ID NO:3) and FIG. 2B (1326258) (SEQ ID NO:5).
In yet another embodiment, the invention provides for alternatively spliced variants of human patched-2 having patched-2 biological activity.
In yet another embodiment, the invention provides for method of using patched-2 for the treatment of disorders which are mediated at least in part by Hedgehog (Hh), especially Desert hedgehog (Dhh). In particular, testicular cancer. In yet another embodiment, the invention provides a method of using antagonists or agonists of patched-2 for treating disorders or creating a desirable physiological condition effected by blocking Hh signaling, especially Dhh signaling. (E.g, contraception).